ABSTRACT Knowing the spectrum of somatic epigenomic changes in cells that give rise to lung cancer may be very important for understanding lung cancer risk. Once translated to non-invasive biomarkers, these newly recognized changes could be crucial for detecting and ameliorating that risk. Recently, next-generation sequencing of epigenome-wide changes and spectra in overt lung cancers, has been reported by our own and other groups. However, the characterization of epigenomic changes in normal, non-clonal epithelial tissue that is at risk or underway for carcinogenesis has not been accomplished. The progenitor basal cells of the bronchial epithelium are by definition long-term residents, and thus may accumulate the epigenetic changes thought to in part underlay the dysregulated state of smoke-induced lung carcinogenesis. Thus, these changes likely distinguish the progenitor elements of the epithelium that are transforming, from that epithelium which is not. We hypothesize that: Those individuals who have a proven propensity for squamous cell lung cancer by virtue of having developed one, have transformed DNA methylome and transcriptome signatures of this propensity in the basal progenitor cells of their cytologically normal bronchial epithelium. Here, based on our prior lung cancer microarray methylome studies, we propose to use a promoter and enhancer-targeted next gen sequencing method for examining visibly normal brushed bronchial specimens yielding basal progenitor cells. These will be provided by former tobacco smoke-exposed individuals with lung cancer versus those without lung cancer of identical age, smoke exposure, COPD status, and underlying lung disease, followed over time to minimize case-control misclassification. In Aim 1, we will generate early primary isolates of stem/progenitor/basal epithelial cells. We will characterize these cells for basal/progenitor and squamous cell cancer marker expression. In Aim 2, we will apply genome-wide next-gen sequencing methods for capture-based bisulfite DNA methylation sequencing assays, as well as next gen microRNA and mRNA transcriptome assays. These landscapes of DNA methylation and transcriptome features will be compared between the basal progenitor cells derived from lung cancer cases versus non- cancer controls. Integration across platforms, technical validation of top hits and functional analysis of a subset of the top hits will be performed. This project will therefore initiate the development of several epigenomic metrics for bronchial epithelial progenitor cells, particularly those likely to undergo carcinogenetic change, as determined by donor phenotype. The comprehensive systems level characterization, rigorous cell phenotypic characterization, and innovative functional evaluation of epigenetic hits, are major notable strengths. The resulting data will be used to develop non-invasive markers of risk in the future, such as sputum or nasal methylation markers, or exhaled microRNA markers, which can be used for targeting early detection and chemoprevention purposes to those individuals most likely to benefit.